The Biochemistry of Malic Acid Metabolism by Wine Yeasts - A Review M. Saayman and M. Viljoen-Bloom1 (1) Department of Microbiology, Stellenbosch University, Private Bag XI, 7602 Matieland, South Africa Submitted for publication: August 2006 Accepted for publication: September 2006 Key words: malic acid; yeast; wine L-Malic acid is an essential intermediate of cell metabolism and the D,L-racemic mixture is used as an acidulant in a variety of foods and beverages. In the wine industry, it plays an important role during grape must fermentation, contributing to the "fixed acidity" that is important. The latter is important in defining the quality of wine. Genetic and biochemical characterisation of the L-malate utilising pathways in several yeast species has indicated that the physiological role and regulation of L-malate metabolism differ significantly between the K(-) and K(+) yeasts. A variety of factors influence the ability of a yeast species to effectively degrade L-malate, including the conditions associated with wine fermentation and the yeast's intrinsic ability to transport and effectively metabolise L-malate inside the cell. This paper reviews the ability of different yeast species associated with grapes and wine to degrade extracellular L-malate, and the underlying mechanisms in the differential utilisation of L-malate by different yeast species. INTRODUCTION Thornton, 1990). Yeast species that are recognised for their abili- The production of good quality wines often requires1 the proper ty to metabolise extracellular L-malate fall into either the K(-) or adjustment of wine acidity in relation to the other wine compo- K(+) yeast groups, depending on their ability to utilise L-malate nents to create a well-balanced bottled product. The different and other tricarboxylic acid (TCA) cycle intermediates as sole chemical and biological factors that contribute to the presence, carbon or energy source (reviewed in Volschenk et al, 2003). The role and degradation of L-malate during wine fermentation have K(+) group includes Candida sphaerica, C. utilis, H. anomala, P. been discussed in detail by Volschenk et al. (2006). The tradi- stipitis and K. marxianus, which all have the ability to utilise tional method to deacidify wine involves the conversion of L- TCA cycle intermediates as sole carbon sources. malic acid to L-lactic acid and CO2 during malolactic fermenta- The K(-) group of yeasts comprises those yeasts capable of util- tion by strains of Oenococcus oeni. The complexities associated ising TCA cycle intermediates only in the presence of glucose or with traditional malolactic fermentation in wine, however, neces- other assimilable carbon sources. According to this definition, S. sitate alternative approaches to reduce wine acidity. One of the cerevisiae, S. pombe, S. pombe var. malidevorans and Z bailii are options is biological deacidification with yeast strains that are all classified as K(-) yeasts (Volschenk et al, 2003). Although able to degrade excess malic acid present in the grape must. grouped together, the K(-) yeasts display significant differences However, not all yeasts that are able to degrade extracellular in their ability to degrade L-malate. The yeast 5. cerevisiae is malic acid are able to survive the conditions associated with regarded as a poor metaboliser of extracellular malate, which has grapes, must and wine, and some may even be considered respon- been attributed to the lack of a mediated transport system for the sible for undesirable characteristics in wine. The metabolic pat- acid (Salmon, 1987). Strains of S. pombe and Z bailii can terns in yeasts also differ significantly, with varying levels of sen- degrade high concentrations of L-malate, but only if glucose or sitivity to the levels of oxygen and glucose. For example, the another assimilable carbon source is present (Baranowski and degradation of extracellular L-malate in some yeasts is subject to Radler, 1984; Rodriquez and Thornton, 1989). strong substrate induction and carbon repression, whereas other Genetic and biochemical characterisation of the L-malate utilis- genera require the presence of glucose or a similar carbon source ing pathways in several yeast species indicates that the physiolog- for the degradation of L-malate. ical role and regulation of L-malate metabolism differs signifi- The degradation of L-malate has been studied in detail in only cantly between the K(-) and K(+) yeasts. The factors involved a few yeast species, including S. cerevisiae (Boles et al, 1998; could include the substrate affinity of the malic enzyme, the rate Volschenk et al., 2003), S. pombe (Osothsilp, 1987; Subden et al, of transport of L-malate into the cell, and other stimulatory or 1998; Viljoen et al., 1994; 1998; 1999), Candida utilis (Cassio inhibitory influences exerted by glucose, fructose or malic acid and Leao, 1993, Saayman et al, 2000; 2006), with limited inves- (Gao and Fleet, 1995). In general, L-malate metabolism in K(-) tigations done in Hansenula anomala (Pichia anomala) (Corte- yeasts is characterised by the absence of glucose repression or Real and Leao, 1990; Amador et al, 1996), Pichia stipitis substrate induction. In contrast, the regulation of L-malate metab- (Thornton and Rodriques, 1996) and Kluyveromyces marxianus olism in K(+) yeasts typically exhibits strong glucose repression (Queiros etal, 1998), Z bailii (Kuczynski and Radler, 1982) and together with substrate induction (Corte-Real and Leao, 1990; Schizosaccharomyces pombe van malidevorans (Rodriques and Amador etal, 1996; Cassio and Leao, 1993; Queiros etal, 1998). Corresponding author: e-mail: [email protected] [Tel: +27-21-808 5859; Fax: +27-21-808 5846] Acknowledgements: Mr. G Coetzee for assistance in preparing the manuscript. S. Afr. J. Enol. Vitic, Vol. 27, No. 2, 2006 113 114 Malic acid degradation in yeast Yeasts that dominate on grapes include species of Rhodotorula, cells, followed by a discussion of the degradation of L-malate in Cryptococcus, Candida, Hanseniaspora (anamorph Kloeckera) S. cerevisiae, S. pombe and C. utilis, with specific reference to the and Mitchnikowia (Fleet, 2003; Jolly et al., 2006). Damaged grapes transport of L-malate and the structure, function and regulation of have shown an occurrence of Hanseniaspora, Candida and the malic enzyme in different yeast species. Metchnikowia species, together with species of Saccharomyces and CENTRAL ROLE OF MALIC ACID IN YEAST METABOLISM Zygosaccharomyces. However, Saccharomyces cerevisiae, the principle wine yeast, is not prevalent on grapes, which may be L-Malate plays a pivotal role in the metabolism of C3 and C4 com- ascribed to the surface chemistry of the grape, tolerance to natural pounds in different subcellular compartments in the yeast cell. stresses and chemical inhibitors, and interactions with other bacte- Depending on the cellular requirements, malate can be oxidised, rial and fungal species (Fleet, 2003). During grape juice fermenta- dehydrated or decarboxylated (see Fig. 1). The oxidation of L- tion, species of Hanseniaspora (Kloeckera), Candida and malate provides oxaloacetate for the turnover of the TCA cycle or Mitchnikowia initiate the fermentation, with Pichia, Issatchenkia for gluconeogenesis via phosphoenolpyruvate (PEP), while the and Kluyveromyces species often present as well. Growth of these decarboxylation of L-malate provides pyruvate for amino acid species declines by mid-fermentation when Saccharomyces cere- and other biosynthetic pathways. Synthesis of L-malate via visiae becomes dominant, mostly due to their ethanol sensitivity malate synthase in the glyoxalate cycle provides a mechanism for (limited to 5-7%). Schizosaccharomyces pombe, Zygosaccharo- the combination of two C2 molecules (glyoxalate and acetyl- myces bailii and Zygosaccharomyces fermentati are well known for CoA) to form a C4 molecule (Fraenkel, 1982). their tolerance of high ethanol concentrations (>10%), but are often The TCA cycle functions primarily in mitochondria where it out-competed by other wine yeasts due to their slow growth. allows for the complete degradation of pyruvate produced during A variety of yeasts, bacteria and filamentous fungi contribute to glycolysis (see Fig. 1). The TCA cycle is only functional under the microbial ecology of grapes and wine and to the chemical aerobic conditions and is required for oxidative growth on pyru- composition of wine, with yeasts having the dominant influence vate, lactate, acetate and ethanol (Boulton et al., 1996). The cycle due to their role in alcoholic fermentation. However, our under- allows for the metabolic flow of carbon between various meta- standing of the different factors involved in the degradation of L- bolic pathways and is a major source of NADH for the produc- malate by yeasts associated with grapes and wine is limited and tion of ATP via oxidative phosphorylation. Some of the enzymes requires further investigation. The following discussion will first of the TCA cycle are also present in the cytosol and peroxisome provide an overview of general L-malate metabolism in yeast where they catalyse similar reactions. Under anaerobic conditions FIGURE 1 L-Malate plays a pivotal role in yeast metabolism. Dashed arrows represent reactions that do not operate during growth on glucose. S. Afr. J. Enol. Vitic, Vol. 27, No. 2, 2006 Malic acid degradation in yeast 115 and in the presence of high concentrations of glucose, cells of S. During fermentative sugar metabolism in yeast, pyruvate, an cerevisiae do not have functional mitochondria